Journal of Cell Science 108, 1143-1153 (1995) 1143 Printed in Great Britain © The Company of Biologists Limited 1995 p80-coilin: a component of coiled bodies and interchromatin granule- associated zones

Francine Puvion-Dutilleul1,*, Sylvie Besse1, Edward K. L. Chan2, Eng M. Tan2 and Edmond Puvion1 1Laboratoire de Biologie et Ultrastructure du Noyau de l’UPR 9044 CNRS, BP 8, F-94801 Villejuif Cedex, France 2The Scripps Research Institute, W. M. Keck Autoimmune Disease Center, 10666 N. Torrey Pines Road, La Jolla, California 92037, USA *Author for correspondence

SUMMARY

We investigated at the electron microscope level the fate of the clusters of interchromatin granules and their associated the three intranuclear structures known to accumulate zones, which were all easily recognizable within the snRNPs, and which correspond to the punctuate immuno- residual nuclear ribonucleoprotein network, was unmodi- fluorescent staining pattern (the coiled bodies, the clusters fied. The data indicate, therefore, that the loosening of interchromatin granules and the interchromatin procedure as well as the high salt extraction procedure granule-associated zones) after exposure to either a low salt preserve the snRNA content of all three spliceosome medium which induces a loosening and partial spreading component-accumulation sites and reveal that interchro- of nucleoprotein fibers or a high ionic strength salt medium matin granule-associated zones are elements of the nuclear and subsequent DNase I digestion, in order to obtain DNA- matrix. The p80-coilin content of coiled bodies was also depleted nuclear matrices. The loosened clusters of inter- preserved whatever the salt treatment used. An intriguing chromatin granules and the coiled bodies could no longer new finding is the detection of abundant p80-coilin within be distinguished from surrounding nucleoprotein fibers the interchromatin granule-associated zones, both before solely by their structure, but constituents of the clusters of and after either low or high salt treatment of cells. interchromatin granules could be detected by in situ Therefore, p80-coilin is an integral constituent of the inter- hybridization with both U1 and U2 DNA probes, and con- chromatin granule-associated zones. stituents of the coiled bodies were detectable mainly with the U2 DNA probe. The interchromatin granule-associated zones, the electron-opacity and compactness of which were Key words: coiled body, electron microscope, HeLa cell, in situ preserved despite the loosening treatment, remained hybridization, interchromatin granule, interchromatin granule- labeled with the U1 DNA probe only. In DNA-depleted associated zone, mild loosening procedure, nuclear matrix, p80- nuclear matrices, the snRNA content of the coiled bodies, coilin, spliceosome, U1 RNA, U2 RNA

INTRODUCTION former and accumulate in considerable amounts in the latter. Both snRNAs accumulate within the clusters of interchro- The components of the cell nucleus involved in transcription matin granules (Spector, 1993; Visa et al., 1993a). As already and splicing have been clearly identified by immunocyto- mentioned (Visa et al., 1993a), the interchromatin granule- chemical and in situ hybridization procedures designed for associated zones, might be equivalent to the A snurposomes electron microscopy. It was clearly shown that the lower-defi- described in amphibian germinal vesicles by the Gall’s group nition speckled pattern observed by fluorescence microscopy (Gall, 1991; Wu et al., 1991). On the other hand, the very low following specific detection of spliceosome components level of labeling of the clusters of interchromatin granules on (Carmo-Fonseca et al., 1991, 1992; Huang and Spector, 1992; electron microscope autoradiographs following labeling of Lamond and Carmo-Fonseca, 1993a,b) corresponds to cells with radioactive uridine (Fakan, 1978; Fakan and perichromatin fibrils and to three different, well-defined struc- Puvion, 1980; Puvion and Moyne, 1981), as well as the total tures, i.e. the clusters of interchromatin granules, the inter- absence of DNA within these structures (Thiry, 1993; Visa et chromatin granule-associated zones and the coiled bodies al., 1993a), exclude the possibility that they could be sites of (Visa et al., 1993a). Although all three structures accumulate transcription and active splicing. Instead, it was proposed that spliceosome components, they differ in their specific con- they are more likely to be concerned with pre- and/or post- tents. U1 snRNAs are present in large amounts in the inter- splicing events such as the final maturation or storage of chromatin granule-associated zones but are much rarer in the snRNP particles, assembly of spliceosomes and/or intron coiled bodies. Conversely, U2 snRNAs are absent in the degradation (Lamond et al., 1990; Visa et al., 1993a,b). How-

1144 F. Puvion-Dutilleul and others ever, the permanent presence of poly(A)+ RNA in clusters of general organization of the clusters of interchromatin granules interchromatin granules (Visa et al., 1993b) and, at least in and their associated zones, whereas that of coiled bodies was certain metabolic conditions, of ribosomal RNA (Puvion et more profoundly changed. On the other hand, the high salt al., 1984b) suggests that they also could be sites of some sort- extraction of cells was without effect on the aspect (and ing and/or degradation of mature RNA molecules. Concerning snRNA content) of the three spliceosome component-accumu- coiled bodies, recent results have shown that this type of lation sites. An unexpected result is that anti-p80 coilin nuclear body could also be multifunctional and related to pre- antibody not only labeled coiled bodies of high salt-extracted, and/or post-splicing events and to unknown nucleolus-associ- loosened and unloosened materials, but also significantly ated functions (Brasch and Ochs, 1992; Lamond and Carmo- labeled the interchromatin granule-associated zones. Fonseca, 1993b; Malatesta et al., 1994). In fact, it is now gen- erally accepted that the perichromatin fibrils represent the MATERIALS AND METHODS main morphological substrate of splicing (Nash et al., 1975; Fakan et al., 1976; Puvion and Moyne, 1978; Puvion and Cell preparation Viron, 1981; Gallinaro et al., 1983). These fibrils form at tran- For electron microscope study, HeLa cells (5×105 cells per 5 cm scription sites, contain nascent RNA (Nash et al., 1975; Fakan plastic culture dish) were grown at 37¡C, in the presence of 5% CO2, et al., 1976; Puvion and Moyne, 1978; Puvion and Viron, in Eagle’s minimum essential medium supplemented with 5% calf 1981; Fakan, 1994) and are associated with hnRNP core pro- serum. Twenty-four hours later, when cells were near confluence, they teins and spliceosome components (Fakan et al., 1984; Puvion were processed for electron microscope study. et al., 1984a; Visa et al., 1993a). This is consistent with the For the observation of unloosened nucleoproteins, cell cultures detection of snRNP antigens on transcripts which are still were fixed in situ, at 4¡C, in 4% formaldehyde (Merck, Darmstadt, attached to the DNA matrix as seen in studies of spread tran- Germany) in 0.1 M Sörensen phosphate buffer, pH 7.3. During the 1 hour fixation, the cells were detached from the culture dish and cen- scription complexes (Fakan et al., 1986) and in observations trifuged. The pellets were dehydrated in increasing concentrations of indicating that splicing may occur co-transcriptionally (Beyer methanol and embedded in Lowicryl K4M (Chemische Werke Lowi, and Osheim, 1988; Wu et al., 1991). Waldkraiburg, Germany). Polymerization was carried out for 5 days Several years ago, we developed a new fixation procedure at −30°C under long wavelength UV light (Philips fluorescence tubes which helped to bridge the gap between the molecular organ- TL 6W) (Roth, 1989). Ultrathin sections were collected on ization of chromatin-RNP complexes in vitro after spreading Formvar/carbon-coated gold grids (200 mesh). of the nuclear contents, on the one hand, and the more compact For the observation of loosened nucleoproteins, culture medium was in situ structures observed in thin sections of routinely fixed removed and cell cultures were exposed for about 5 seconds to distilled cells, on the other hand (Puvion-Dutilleul and Puvion, 1980; water adjusted to pH 8.5 with 0.1 M borate buffer (Puvion-Dutilleul Puvion-Dutilleul, 1983). This method, adapted from Miller’s and Puvion, 1980). The cells were then covered for 1 hour with 6 ml of spreading technique (Miller and Beatty, 1969), consists of 1% formaldehyde aqueous solution containing 0.4% Photo flo (a com- mercial wetting agent containing Triton X-100; Kodak-Pathé, Chalon, preparation of cells in a detergent-containing hypotonic France), and 0.1 M sucrose. This solution was previously adjusted to fixative solution, which induces only a mild loosening of pH 8.5 with the borate buffer. During the 1 hour loosening treatment, nucleoproteins instead of the extensive dispersal of chromatin the cells were scraped and centrifuged. The resulting translucent pel- employed by Miller. Until now, our modification of Miller’s lets were dehydrated in methanol and embedded in Lowicryl as procedure has been used for studying in situ transcription described above for pellets of unloosened cells. complexes in normal cells (Puvion-Dutilleul and Puvion, 1980; Nuclear matrices of HeLa cells were kindly provided by F. Harper Vazquez-Nin et al., 1989, 1992), drug-treated cells (Puvion et (Villejuif, France). They were prepared from cells still attached to the al., 1981; Puvion-Dutilleul and Puvion, 1981) and virus- plastic culture dishes as previously described (Puvion et al., 1988) infected cells (Puvion-Dutilleul et al., 1980; Puvion-Dutilleul according to a slight modification of the method of Buckler-White et and Puvion, 1982), and for studying the insertion of viral al. (1980). Briefly, cultures were treated for 3 minutes at 4¡C in TMS genomes into intranuclear viral capsids (Puvion-Dutilleul et buffer (0.25 M sucrose, 5 mM MgSO4, 50 mM Tris-HCl, pH 7.4) con- taining 1% Triton X-100 prior to being incubated for 30 minutes at al., 1989). However, the three structures which accumulate 37¡C in TMS buffer containing 50 µg/ml DNase I (pancreatic DNase spliceosome components have still not been identified after I type IV, Sigma St Louis, MO, USA). After washing with 10 mM loosening and spreading of nuclear constituents. Tris-HCl buffer, pH 7.4, plus 0.2 MgSO4, cultures were treated for In this study, we took advantage of the combined procedures 2× 15 minutes with a high salt buffer (2 M NaCl, 0.2 mM MgSO4, of electron microscopic in situ hybridization and of immun- 10 mM Tris-HCl, pH 7.4) and washed in 10 mM Tris-HCl buffer odetection to study the effects of moderate decondensation of added with 0.2 mM MgSO4 and fixed with formaldehyde. During the chromatin on the accumulation sites of spliceosome com- fixation, matrices were detached from their substratum and cen- ponents. It should be noted here that even moderate decon- trifuged. The resulting pellets were dehydrated in methanol and densation of chromatin can hamper purely morphological iden- embedded in Lowicryl K4M as above. tification of nuclear constituents, but its combination with in For optical study, cells were grown on glass coverslips. Some situ hybridization and immunodetection provides a labeling of cultures were fixed with cold methanol-acetone (3:7, v/v) for 5 minutes at −20¡C and air-dried, whereas others were fixed for 20 the constituents that are being studied. The results were minutes with 4% formaldehyde in PBS, washed for 15 minutes in compared with the fate of spliceosome component-accumula- PBS, incubated for 15 minutes in 0.30% Triton X-100 in PBS and tion sites in DNA-depleted nuclear matrices. washed again in PBS prior to being used for immunolabeling and in U1 and U2 snRNAs were hybridized to biotinylated specific situ hybridization. genomic probes, and coiled bodies were identified by their labeling with anti-p80 coilin antibody. Our results reveal a Immunodetection of p80-coilin somewhat mild effect of our loosening procedure on the For in situ detection at the ultrastructural level of structures contain-

p80-coilin in HeLa cell nuclei 1145 ing p80-coilin, grids bearing Lowicryl sections of HeLa cells, with or minutes at 64¡C on 2 µl hybridization solutions containing either U1 without loosening of the nucleoproteins, and nuclear matrices were or U2 biotinylated DNA probe, rapidly rinsed over 20 µl drops of cold incubated for 30 minutes at room temperature on a 10 µl drop of rabbit PBS and transfered over drops of BSA (5% in PBS) for 3 minutes as serum R288 containing polyclonal antibodies against recombinant previously described (Besse and Puvion-Dutilleul, 1994). A rabbit p80-coilin (Andrade et al., 1993), diluted 1/50 in PBS. After 15 anti-biotin IgG antibody (1/75 in PBS) (Enzo Biochem. New York, minutes washing in PBS, the grids were floated for 30 minutes on a N Y, USA) was used as the primary antibody. A rhodamine TRITC- 10 µl drop of 1/25 dilution of goat anti-rabbit IgG-conjugated to gold conjugated goat anti-rabbit IgG (Nordic) diluted 1/50 in PBS was used particles, 10 nm in diameter (Biocell Research Laboratories, Cardiff, as the secondary antibody. Samples were mounted in glycerol-PBS UK). After a final PBS wash, the grids were rapidly rinsed in a jet of and observed using an Axiophot microscope. distilled water, air-dried and stained for 10 minutes with 5% aqueous uranyl acetate. For controls, a 1/10 dilution of normal rabbit serum was used instead of serum R288. Grids were observed after a 10- RESULTS minute uranyl acetate staining. Immunogold localization of p80-coilin For immunofluorescence detection of p80-coilin, drops of 10 µl of serum R288 and normal rabbit serum, both diluted 1/50 in PBS, were In conventionally formaldehyde-fixed HeLa cells, gold deposited at the center of the coverslips bearing either methanol- particles which localized p80-coilin accumulated mainly over acetone-fixed cells or formaldehyde-Triton-treated cells. Incubations the coiled bodies, 0.5 to 1 µm in diameter (Fig. 1A). Higher were performed for 30 minutes at 37¡C in a wet chamber. After three magnifications clearly revealed the tight association of the washings in PBS, the coverslips were incubated for 30 minutes at label with the coiled threads of about 20 nm in diameter and room temperature in rhodamine (TRITC)-conjugated goat anti-rabbit the total absence of labeling in the interstices between the IgG (Nordic Immunology, Tilburg, The Netherlands), 1/50 in PBS. threads (Fig. 1B). In addition, cross-sections of the coiled Following 3×5 min washings in PBS, the coverslips were finally rinsed in distilled water and mounted in glycerol-PBS. Samples were threads had the appearance of rings surrounding electron- observed using an Axiophot microscope (Carl Zeiss, Oberkochen, translucent cores, which suggests that coiled bodies might Germany) equipped with epifluorescence. consist either of hollow threads or of threads composed of more than one constituent. Gold particles also were present Localization of U1 and U2 snRNAs by in situ hybridization over compact fibrillar masses, which we named the interchro- The two biotinylated probes used in this study were kindly provided matin granule-associated zones (Visa et al., 1993a) (Fig. by J. P. Bachellerie and have been described in our previous publi- 1A,C). These latter structures were sparsely and irregularly cations (Visa et al., 1993a; Puvion-Dutilleul et al., 1994). Briefly, the labeled. The contiguous clusters of interchromatin granules U1 DNA probe (kindly provided by J. E. Dahlberg) corresponds to a were always devoid of labeling. A few gold particles were 260 bp BglII fragment of human DNA encompassing the entire U1 RNA coding region, which was cloned into the vector pSP64. The U2 dispersed over the surrounding nucleoplasm. DNA probe (kindly provided by T. Pederson) corresponds to the In loosened nucleoproteins following fixation of the cells in a pSPU2pre recombinant plasmid in which the entire U2 RNA coding hypotonic solution of formaldehyde, the nuclei were swollen sequence plus 12 3′-flanking nucleotides were cloned into the vector and the chromatin separated into threads which filled the nucle- pSP64. Both DNA probes were biotinylated by nick-translation of the oplasm. The chromatin fibers of Lowicryl-embedded material whole plasmid using biotinylated dATP (biotin 14-dATP, Bethesda are about 12-15 nm wide; that is, thinner than those in Epon- Research Lab., Bethesda, MD/USA). embedded material (20-25 nm wide; Puvion-Dutilleul and In order to localize U1 and U2 snRNAs by electron microscope in Puvion, 1980). These fibers were punctuated irregularly by situ hybridization, gold grids bearing sections of formaldehyde-fixed granules which had grossly the same diameter as the fibers and cells, loosened nucleoproteins or nuclear matrices were floated on 1- 2 µl of hybridization solutions containing 50% deionized formamide, corresponded to chromatin fibers seen in cross-section. The 10% dextran sulphate, 2× SSC buffer (1× SSC = 0.15 M NaCl, 0.015 clusters of interchromatin granules and the coiled bodies were M sodium citrate), 10 µg/ml biotinylated probe, and 400 µg/ml com- no longer identifiable by morphological criteria because their petitor Escherichia coli DNA. The hybridization solutions were fibers were thinner and resembled those of the surrounding treated for 4 minutes in boiling water in order to denature the double- spread nucleoprotein fibers. Only the interchromatin granule- stranded DNA (dsDNA) molecules and rapidly chilled prior to use. associated zones, the compact fibrillar organization of which Hybridizations with each hybridization solution were performed in a was preserved by the loosening treatment, were detectable, and wet chamber for 3.5 hours at 64¡C, i.e. in experimental conditions they could be identified even in the absence of specific labeling. which were established in our previous studies (Visa et al., 1993a). They also could be decorated by gold particles (Fig. 2A) Hybrids formed at the surface of the Lowicryl sections were revealed by incubating the grids for 30 minutes on a 5 µl drop of anti-biotin whereas adjacent areas which might correspond to the clusters antibody conjugated to gold particles, 10 nm in diameter (Biocell of interchromatin granules were entirely devoid of labeling. Research Lab., Cardiff, UK), diluted 1/25 in PBS. Grids were Gold particles bound to p80-coilin also were abundant over observed after a 10 minute staining with 5% aqueous uranyl acetate. roughly spherical areas, 0.5 to 1 µm in diameter, which We verified that a protease pre-treatment of sections (0.2 mg/ml consisted of 12 nm thick coiled filaments (Fig. 2B). These protease from Streptomyces griseus type VI; Sigma, St Louis, MO, labeled areas were coiled bodies, the diameter of the filaments USA) in distilled water for 15 minutes at 37¡C) did not markedly having been markedly decreased under the loosening con- improve the accessibily of targets to the probes. In addition, the speci- ditions. Once again, labeling was associated exclusively with ficity of the labeling was attested by the total absence of labeling the filaments of the coiled bodies whereas the surrounding following an RNase treatment of sections prior to hybridization (1 mg/ml RNase A from bovine pancreas; BDH Biochemicals, Poole, nuclear material was decorated with only a few randomly UK) in 10 mM Tris-HCl, pH 7.3, for 1 hour at 37¡C). dispersed gold particles. For the detection of U1 and U2 snRNA at the optical level, cover- In nuclear matrices following both DNase and high salt slips previously treated with formaldehyde-Triton were cut up into extractions prior to fixation, the clusters of interchromatin gran- little squares (less than 10 mm side). These were floated for 90 ules and their associated zones were not modified by the treat- 1146 F. Puvion-Dutilleul and others

Fig. 1. Immunogold labeling of Lowicryl sections of formaldehyde-fixed HeLa cells using anti-p80 coilin antibody. Uranyl acetate staining. (A) Low magnification of a part of a nucleus. Gold particles mainly accumulate over the coiled body (cb) and the two interchromatin granule- associated zones (stars) whereas the clusters of interchromatin granules (ig) are devoid of labeling. A few gold particles are scattered over the surrounding nucleoplasm. (B) Higher magnification of a coiled body (cb), which shows the exclusive labeling of its filaments, about 20 nm in diameter, without labeling of the interstitial spaces. The arrow points to a ring-shaped configuration of a cross-sectioned filament. (C) Higher magnification of an interchromatin granule-associated zone (star) which shows an intense although uneven labeling. Bars, 0.5 µm. ments and were easily identifiable among the residual ribonu- homogeneous, moderately electron-opaque matrix. The cleoprotein network. Coiled bodies, which were recognizable immunogold method revealed an intense labeling restricted to by their round shape, consisted of filaments enclosed within a the coiled bodies (Fig. 2C) and the interchromatin granule-asso- p80-coilin in HeLa cell nuclei 1147

Fig. 2. Immunogold labeling of Lowicryl sections of mildly loosened nucleoproteins (A,B) and nuclear matrices (C,D) of HeLa cells using anti-p80 coilin antibody. Uranyl acetate staining. (A) The compact fibrillar organization of the interchromatin granule-associated zone (star) is still detectable despite the mild loosening treatment of the cell. Its associated cluster of interchromatin granules is no longer identifiable among the spread nucleoprotein fibers. Gold particles are mainly found over the interchromatin granule-associated zone. (B) The loosened coiled body (cb) consists of a loose network of thin, intensely labeled filaments. (C) In a DNA-depleted nucleus, the easily recognizable coiled body (cb) is intensely labeled. (D) The interchromatin granule-associated zone (star) observed in a nuclear matrix is intensely labeled and juxtaposed to an unlabeled cluster of interchromatin granules (ig). Bars, 0.5 µm. ciated zones whereas their still adjacent clusters of interchro- Immunofluorescence localization of p80-coilin matin granules were entirely devoid of labeling (Fig. 2D). Since electron microscopic detection of p80-coilin by the use No labeling was obtained when normal rabbit serum was of serum R288 revealed the presence of the protein not only in used instead of serum R288. the coiled bodies but also in the interchromatin granule-asso- 1148 F. Puvion-Dutilleul and others ciated zones, we have reinvestigated the intranuclear distribu- formaldehyde/Triton-treated cells, the extranucleolar region of tion of p80-coilin at the optical level. Following either the nuclei showed a diffuse staining in which more bright spots methanol-acetone fixation or formaldehyde-Triton treatment of were observed. Following the use of the U2 DNA probe (Fig. cells, we observed that serum R288 gave 1 to 6 fluorescent 3F), the general nucleoplasmic labeling is less intense, spots in most of the nuclei whatever the fixative used (Fig. rendering bright patches more visible. 3A,C). About 10% of nuclei also displayed elongated fluores- cent material which had the appearance of short filaments (Fig. 3B,D). DISCUSSION

Localization of U1 and U2 RNA by electron In previous publications (Visa et al., 1993a; Puvion-Dutilleul microscope in situ hybridization et al., 1994) we have described the results of studies identify- Denatured biotinylated U1 and U2 DNA probes were applied ing those intranuclear structures of HeLa cells which are accu- to sections of unloosened and loosened HeLa cells and nuclear mulation sites for spliceosome components. In these reports, matrices in order to hybridize them to complementary RNA we presented evidence that, in addition to the well-known sequences, and the hybrids were subsequently revealed by coiled bodies and clusters of interchromatin granules, a third direct immunogold labeling. Data with conventionally fixed structure concomitantly accumulates snRNP. This new cells were previously reported (Visa et al., 1993a); therefore, structure was designated the interchromatin granule-associated the scope of the present study was restricted to the distribution zone because of its usual contiguity with the clusters of inter- of U1 and U2 snRNAs in loosened nucleoproteins and nuclear chromatin granules (Visa et al., 1993a). In addition, we matrices. observed that the distribution of spliceosome components is As previously reported (Visa et al., 1993a), after hybridiz- dynamic and depends upon the physiological state of the cell, ation of U1 and U2 DNA probes to sections of cells conven- since only one type of spliceosome-component accumulation tionally fixed with formaldehyde, gold particles were found site persists in cells infected with either adenovirus (Puvion- over the perichromatin fibrils and the clusters of interchromatin Dutilleul et al., 1994) or herpes simplex virus (Besse et al., granules. The interchromatin granule-associated zones were 1994) when the replicative activity of viral genomes becomes labeled with the U1 DNA probe only. The coiled bodies were intense. In the current investigation, we extend our earlier labeled intensely with the U2 DNA probe and only slightly observations with evidence that all three spliceosome- with the U1 DNA probe. component accumulation sites are morphologically stable Following mild loosening of nuclear constituents, the inter- structures which persist without changes in their snRNA chromatin granule-associated zones, which were easily identi- contents in nuclear matrices, i.e. following a high salt treatment fiable among the loosened chromatin fibers, were labeled with of cells, as well as following experimentally induced mild the U1 DNA probe (Fig. 4A) but were entirely devoid of loosening of nucleoproteins by a low salt treatment which labeling by the U2 DNA probe. Both probes, however, labeled spreads the nucleoproteins enough to allow a clear in situ visu- areas of irregular shape which consisted of granules, 13 to 15 alization of actively transcribed genes (Puvion-Dutilleul and nm in diameter, inter-connected by low contrasted filaments of Puvion, 1980; Puvion-Dutilleul, 1993). In order to facilitate the about 5 nm in width, thereby forming a continuous and slack observation of coiled bodies among the residual ribonucleo- network. Only the intense labeling of these loose networks, protein network and the partially spread nucleoproteins, we which corresponded to the clusters of interchromatin granules, used the anti-p80-coilin antibody as a marker (Andrade et al., allowed their clear delineation from the surrounding innumer- 1993; Carmo-Fonseca et al., 1993; Lamond and Carmo- able chromatin fibers. Labeled clusters of interchromatin Fonseca, 1993b) and discovered that p80-coilin also was granules were observed both adjacent to (Fig. 4A) and at a con- present in the interchromatin granule-associated zones, not siderable distance from (Fig. 4B) the interchromatin granule- only in untreated cells but also after high or low salt treatment, associated zones. Coiled bodies were recognizable only as well as in the coiled bodies. following the use of the U2 DNA probe, which intensely The most intriguing finding of this study is the binding of labeled small, clearly delineated tangles of coiled filaments anti-p80-coilin antibody to the interchromatin granule-associ- (Fig. 4C). Gold particles were directly associated with the latter ated zones as well as the coiled bodies. Indeed, the anti p80- filaments and never with their interstices. After the use of the coilin antibody decorated the interchromatin granule-associ- U1 DNA probe, the coiled bodies were difficult to distinguish ated zones intensely. Thus electron microscopy reveals that the among the loosened chromatin fibers because of their minimal location of p80-coilin is not restricted to the coiled bodies, level of labeling. since anti-p80-coilin antibody binds to another structure which In nuclear matrices, the clusters of interchromatin granules is different in shape (round, ovoid or cylindrical), appearance remained labeled with the U1 (Fig. 5A) and U2 DNA probes (compact and fibrillar) and snRNA composition (devoid of U2 (Fig. 5B). Once again, their associated zones were labeled with snRNA). These electron microscope data are reinforced by the U1 DNA probe (Fig. 5A) and never with the U2 DNA immunofluorescence data which demonstrate the binding of probe (Fig. 5B). Conversely, coiled bodies were intensely anti-p80-coilin antibody over two morphologically distinct labeled with the U2 DNA probe and more slightly with the U1 structures, i.e. spherical and cylindrical structures. In addition, DNA probe (Fig. 5B,C). fluorescence microscopy revealed that only 10% of the nuclei contain labeled elongated structures in addition to bright spots, Immunofluorescence labeling of U1 and U2 snRNAs while labeled interchromatin granule-associated zones by optical in situ hybridization observed with the electron microscope were present in about Following in situ hybridization of U1 DNA probe (Fig. 3E) on 50% of the sectioned nuclei. Taken together, these observa- p80-coilin in HeLa cell nuclei 1149

Fig. 3. Indirect immunofluorescence localization of p80-coilin (A-D) and U1 snRNA (E) and U2 snRNA (F) in methanol-acetone-fixed (A,B) and formaldehyde-Triton-treated (C-F) HeLa cell cultures. (A-D) Localization of p80-coilin. The specific antibodies predominantly stain coiled bodies, which appear as bright nuclear foci (arrowheads). The p80-coilin-containing intranuclear structures in some nuclei, however, have the aspect of elongated, convoluted, bright filaments (arrows). (E,F) Localization of U1 and U2 snRNAs by in situ hybridization on formaldehyde/Triton-treated cells. Following the use of the U1 DNA probe (E), a diffuse nucleoplasmic fluorescence is observed in which more bright spots are present. Bright patches only are observed following the use of U2 DNA probe (F). Bars, 10 µm. tions confirm that the interchromatin granule-associated zones of previous study by Carmo-Fonseca et al. (1993) showing that are pleomorphic structures and reveal that the rounded ones are coiled bodies are kinetic structures and that their steady-state part of the spotted fluorescence. This is interesting in the light size changes depending upon the metabolic activity of the cell, 1150 F. Puvion-Dutilleul and others

Fig. 4. Localization of U1 (A) and U2 (B,C) snRNAs by in situ hybridization of U1 and U2 DNA probes, respectively. Lowicryl sections of mildly loosened nucleoproteins of HeLa cells. Uranyl acetate staining. (A) Localization of U1 snRNA. Gold particles labeled the interchromatin granule-associated zone (star) and the cluster of interchromatin granules (ig). A few gold particles are scattered over the surrounding loosened nucleoproteins. (B) Localization of U2 snRNA. Gold particles are mainly present over the round cluster of interchromatin granules (ig). (C) Localization of U2 snRNA. Gold particles are intensely accumulated over the filaments of the coiled body (cb). Bars, 0.5 µm. whereas the intracellular level of p80-coilin remains stable, Variations in the ionic strength affected the aspect of coiled which implies that p80-coilin must exist outside the coiled bodies only. After a high salt treatment of cells in order to bodies. obtain nuclear matrices, coiled bodies appear to consist of thin p80-coilin in HeLa cell nuclei 1151

Fig. 5. Localization of U1 (A,C) and U2 (B,D) snRNAs by in situ hybridization of U1 and U2 DNA probes, respectively. Lowicryl sections of nuclear matrices. Uranyl acetate staining. (A,C) Following the use of the U1 DNA probe, gold particles are numerous over the cluster of interchromatin granules (ig) and its associated zones (stars) and rarer over the coiled body (cb). (B,D) Following the use of the U2 DNA probe, gold particles are numerous over the cluster of interchromatin granule (ig) and the coiled body (cb). The interchromatin granule-associated zones (stars) are entirely devoid of labeling. Bars, 0.5 µm.

filaments enclosed in a round, well-delineated, moderately clearly revealed that the low salt treatment did not induce electron-opaque matrix, as seen in Fig. 2C. The loosening swelling of the coiled bodies and did not disperse their procedure markedly modified the appearance of the coiled filaments but simply made them thinner. On the other hand, the bodies by also reducing the diameter of their filaments; shape and fibrillar organization of the interchromatin granule- however, without concomitant formation of a matrix. As seen associated zones were preserved after combined hypotonic in Fig. 2B, following a low salt treatment of cells, the coiled shock and detergent treatment, as well as after combined bodies are indistinguishable morphologically from the sur- DNase digestion and high salt treatment, which renders these rounding loosened nucleoproteins. Only the binding of anti- structures identifiable even in the absence of labeling. The p80-coilin antibody and U2 DNA sequences to round networks association of the interchromatin granule-associated zones of filaments allowed us to identify the coiled bodies, and they with the clusters of interchromatin granules was preserved 1152 F. Puvion-Dutilleul and others whatever the salt treatment used, which reinforces our assump- Comité Départemental du Val-de-Marne de la Ligue Nationale contre tion that the two structures are morphologically and function- le Cancer. ally linked. Taken together, the data demonstrate that the inter- chromatin granule-associated zones, like coiled bodied and interchromatin granules, are elements of the nuclear matrix. In REFERENCES addition, the level of p80-coilin and snRNAs in both coiled bodies and interchromatin granule-associated zones was not Andrade, L. E. C., Tan, E. M. and Chan, E. K. L. 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